A new cell culture protocol for enrichment and genetic modification of adult canine Schwann cells suitable for peripheral nerve tissue engineering

Easily applicable techniques are presented to obtain high numbers of enriched canine Schwann cells (cSC) in a short time-window. The potential of adult SC for tissue engineering of peripheral nerves and ex vivo gene therapy is obvious from physiological events taking place after peripheral nerve transection [Haastert, K., Grothe, C., 2007. Gene therapy in peripheral nerve reconstruction approaches. Curr. Gene Ther. 7, 221-228]. The presented techniques were modified from a protocol for cultivation and expansion of adult cSC by others [Pauls, J., Nolte, C., Forterre, F., Brunnberg, L., 2004. Cultivation and expansion of canine Schwann cells using reexplantation. Berl. Munch. Tierarztl. Wochenschr. 117, 341-352] and own experiences in rodent and human SC cultivation and transfection [Haastert, K., Mauritz, C., Chaturvedi, S., Grothe, C., 2007. Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol. Nat. Protoc. 2, 99-104]. A purity of about 80% cSC achieved by immunopanning techniques and selective culture conditions is 2.5 fold higher as previously reported (Pauls et al., 2004). Additionally, highly enriched cSC populations are available in 3-4 weeks, only half the time period reported previously (Pauls et al., 2004). Furthermore, electroporation and genetic modification of cSC is reported for the first time.     

圈养麇鹿冬季昼间行为及活动规律

2008年11-12月在济南动物园,采用瞬时扫描取样法、焦点动物取样法和全事件取样法,对9只圈养麋鹿的行为及活动规律进行了研究。结果表明,麇鹿冬季行为时间分配比例依次为卧息（36．45％±7．34％）、取食（24．79％±8．11％）、运动（18．67％±5．93％）、反刍（17．50％±5．52％）、其他（2．59％±0．87％）。在冬季麋鹿的卧息在1d内有1个高峰期（12：30～14：30）,取食在1d中有2个高峰期（8：30～9：30和17：30～18：30）,占糜鹿各种行为比例最多的2种行为在1d中是交替出现的,运动在10：30和15：30各有1个较为明显的高峰期,反刍在1d中有1个高峰期（9：30～11：30）,其他行为在1d中发生的较为随机,没有明显的高峰期。年龄因素对糜鹿冬季昼间的卧息、取食和运动行为的影响差异极显著（P〈0．01）,对其他行为影响显著（P〈0．05）,对反刍行为影响不显著（P〉0．05）;性别因素对糜鹿冬季昼间的行为影响不显著（P〉0．05）。     

圆环病毒2型人工感染猪的排毒情况及体内分布规律的初步研究

猪圆环病毒2型（PCV2）是引起断奶仔猪多系统衰竭综合症（PMWS）的重要病原,它给养猪业带来巨大的经济损失。本研究利用PCR及荧光定量PCR检测猪人工感染猪圆环病毒2型后的排毒及体内分布规律。结果表明,感染猪可以通过呼吸道和消化道进行排毒,而且病毒持续存在;PCV2病毒主要存在于病猪的淋巴结、肾脏、肺脏中,在肝脏和脾脏中也少量存在。     

The Yields and Carbon and Nitrogen Balances in the Polyfactorial Field Experiments

The polyfactorial long-term field experiments have been founded in 1979 in several experimental stations located in different soil and climatic conditions, representing a climate and pedosequence of arable soils in the Czech Republic. Four of these experiments exist till the present time. Essentially, the same experimental design that includes different fertilisation with farmyard manure and/or with mineral fertilisers (N, P, K), liming and stand density, each in five levels with four replications, have been applied for all these experiments. Practically, the same eight year crop rotations were applied during the first and second rotations. A conventional tillage has been applied. Organic carbon and nitrogen contents in soil, dry matter of the main and second products, nitrogen uptake by the main and second products and carbon and nitrogen balances have been evaluated in six selected variants of organic and mineral fertilisation over the time period 1996 to 2000.     

Growth and uptake rates of P,K, Ca and Mg in wheat 1

The relation between plant age and nutrient absorption properties of red winter wheat (Triticum aestivum L.) roots were investigated. Understanding the change in ion uptake parameters with increasing plant age is helpful in devising efficient fertilization systems. Such information can be used to determine the nutrient levels needed in the soil to supply nutrients rapidly enough to the root surface to minimize deficiencies. Wheat was grown for periods up to 40 days in solution culture in a controlled climate chamber. Sequential harvest and nutrient influx measurements were made. Shoot growth was exponential with time to 30 days and linear thereafter. Root dry weight increased linearly with time at a slower rate than shoot dry weight. Root length increased linearily with time. With increasing plant age there was a reduction in average P and K uptake rate while average uptake rates for Ca and Mg remained relatively unchanged. With increasing plant age, the maximum influx, I_max. for P and Mg remained constant, but for K and Ca, there was a decrease. For the Michael is constant, Km, no change was observed for P, an increase occurred for K, and a decrease for Ca and Mg, as the wheat plant grew from 5 to 40 days.     

西南某矿区土壤重金属的人体生物有效性及健康风险评估

利用生物可给性修正后土壤重金属经口摄入的人体健康风险值也可能被高估,而应用体内实际摄入剂量（生物有效性）作为健康风险评价指标将更为准确,但是基于生物有效性的矿区土壤重金属污染健康风险评估报道较少。以云南文山某矿区为研究对象,分析了5个土壤样点中镉（Cd）、铅（Pb）、锌（Zn）和铜（Cu）的含量,通过体外消化法测定了4种重金属的生物可给性,并结合BALB/c白变种实验室小鼠实验分析了重金属Cd的相对生物有效性（RBA）,基于此探讨该矿区土壤重金属Cd、Pb、Zn和Cu对人体产生的健康风险。结果表明,本研究区域土壤Cd污染较为严重,含量为2.06 mg·kg-1。四种重金属的生物可给性在胃相中的范围分别为24.29%～50.55%、7.68%～17.87%、24.61%～32.18%、7.75%～37.87%,在肠相中分别下降至22.78%～44.32%、1.64%～5.22%、14.10%～28.11%、8.51%～31.49%。动物实验测得的Cd在肝脏中RBA为1.31%～48.39%,肾脏中为2.83%～8.58%,肝和肾复合器官中为4.60%～50.95%,且肝和肾复合器官是测定...     

The effect of in vivo growth on the cellular and extracellular components of the marine bacterial pathogen Photobacterium damsela subsp. piscicida

Photobacterium damsela subsp. piscicida, the causative agent of fish pasteurellosis, was grown in vivo. Bacterial cells and extracellular products (ECPs) were analysed via electrophoresis and immunoblot analysis, using specific sea bass antisera. Growth in vivo induced the synthesis of unique bacterial cell proteins at > 206, 206, 21.3, 18, 7.6 and < 7.6 kDa. Sea bass serum raised against live bacterial cells of the pathogen and especially a sea bass serum raised against formalin-inactivated bacterial cells grown in a specific novel medium recognized the novel antigens at > 206 (associated with iron sequestration), 21.3, 7.6 and < 7.6 kDa, suggesting that the latter medium conserves the synthesis of natural bacterial cell proteins in vitro. In vivo growth of the pathogen induced the synthesis of more toxic ECPs in comparison with in vitro growth and an inverse correlation between total protein concentration in the ECPs and toxicity per unit of protein was observed. Substrate-polyacrylamide electrophoresis revealed the presence of in vivo synthesized ECPs of the pathogen (proteases) at 175, 132, < 79 and 48.3 kDa. Histological examination of tissues isolated from fish injected with these ECPs revealed inflammatory and necrotic lesions in the spleen, liver, head kidney, intestine and heart as soon as 48 h post-introduction of the ECPs.     

The in vivo incorporation and metabolism of [1-14C] linolenate (18:3n-3) in liver, brain and eyes of juveniles of rainbow trout Oncorhynchus mykiss L and gilthead sea bream Sparus aurata L.

Accumulation of docosahexaenoic acid (DHA; 22:6n-3) in brain and eyes during development has been demonstrated in fish but it is not clear whether liver or neural tissues themselves are of greater importance in the biosynthesis of DHA from dietary 18:3n-3. In the present study, we investigated the in vivo metabolism of intraperitoneally injected [1-¹⁴C]18:3n-3 in liver, brains and eyes of young juvenile fish. Metabolism was followed over a 48h time-course in order to obtain dynamic information that could aid the elucidation of the roles of the different tissues in the biosynthesis and provision of DHA from dietary 18:3n-3. The study was performed in both a freshwater fish, rainbow trout Oncorhynchus mykiss L and a marine fish, gilthead sea bream Sparus aurata L to determine the effect that low or limiting5-desaturase activity may have in this process. As expected, the results showed that although the sea bream incorporated more 18:3n-3 into its lipids, metabolism of the incorporated fatty acid by de saturation and elongation was generally greater in the trout. In liver, the percentages of radioactivity recovered in tetraene and pentaene products were greater in trout than in sea bream although there was no difference in hexaenes. In contrast, the re covery of radioactivity in DHA was significantly greater in brain in trout compared to sea bream. In both species, the percentage of radioactivity recovered in desaturated/elongated products was much lower in liver than in brains and eyes, but that percentage increased over the 48h time-course. In trout though, the highest percentages of desaturated products in brain and eye were observed after 12 and 24h, respectively. However in sea bream the highest percentages of desaturated products in the neural tissues were observed after 24-48h. Radioactivity was recovered in 24:5n-3 and 24:6n-3, intermediates in the 4-independent ("Sprecher shunt") pathway for the synthesis of DHA, in both species, especially in the brain and eyes. Overall, although the results cannot eliminate a role for liver in the biosynthesis and provision of DHA for developing neural tissues in fish, they suggest that DHA can be synthesised in fish brain and eye in vivo.     

Rescue In Vitro Fertilization Method for Legacy Stock of Frozen Mouse Sperm

Sperm cryopreservation has been widely adopted for maintenance of the genetically engineered mouse (GEM). The cryopreserved sperm are being exchanged among many institutes worldwide. However, the recipients are not always able to obtain high fertilization rates with the frozen sperm shipped from senders. In this study, we cryopreserved mouse sperm via various methods and performed in vitro fertilization (IVF) in which the combination of methyl-beta-cyclodextrin for sperm preincubation and reduced glutathione for insemination was used (the MBCD-GSH IVF). In addition, frozen sperm sent from the Jackson Laboratory (USA) were thawed and used for IVF in the same manner. The fertilization rates of both the sperm cryopreserved via the methods applied in some countries and the cryopreserved GEM sperm improved when used with the MBCD-GSH IVF method. Therefore, we strongly believe that the MBCD-GSH IVF method brings about relatively high fertilization rates with any strain of frozen mouse sperm.